Germline TFAM levels regulate mitochondrial DNA copy number and mutant heteroplasmy in C. elegans

The mitochondrial genome (mtDNA) is packaged into discrete protein-DNA complexes called nucleoids. mtDNA packaging factor TFAM (mitochondrial transcription factor-A) promotes nucleoid compaction and is required for mtDNA replication. Here, we investigate how changing TFAM levels affects mtDNA in the Caenorhabditis elegans germ line. We show that increasing germline TFAM activity boosts mtDNA number and significantly increases the relative proportion of a selfish mtDNA mutant, uaDf5 . We conclude that TFAM levels must be tightly controlled to ensure appropriate mtDNA composition in the germ line.


Figure 1. Putative germline TFAM overexpression modulates mtDNA copy number and heteroplasmy:
(A) Schematic of strategies to increase germline TFAM levels via transgenic expression at the endogenous glh-1 locus. Strategy (1) utilizes the viral T2A ribosomal skipping sequence: sequence encoding T2A::TFAM was inserted in frame with the endogenous glh-1 coding sequence, resulting in the production of a single mRNA that is translated into two independent polypeptides via ribosomal skipping of the T2A sequence. Strategy (2) utilizes the C. elegans SL2 trans-splicing recognition sequence of the gene rla-1: sequence encoding SL2::TFAM was inserted immediately following the endogenous glh-1 stop codon, resulting in the production of two independent mRNAs that could then be translated into two polypeptides independently of each other. Endogenous glh-1 sequence (maroon), TFAM(hmg-5) sequence (green), T2A sequence (cyan), , and technical ddPCR replicates in (C), from each of three color-coded biological replicates; the mean from each replicate is shown as a larger circle, the mean of means as a horizonal line, and the S.E.M as error bars. n.s., not significant (p> 0.05), **p ≤ 0.01, **** p ≤ 0.0001, unpaired two-tailed Student's t-test.

Description
The mitochondrial transcription factor-A (TFAM) plays essential roles in regulating mtDNA copy number, compacting nucleoids, and replicating/transcribing mtDNA (Garrido et al. 2003, Lewis et al. 2016, Fu et al. 2020. Global reduction of TFAM activity has a conserved effect on mtDNA copy number in metazoans: genetic knockdown severely reduces mtDNA levels in mammals, fish, flies, and cell culture systems (Larsson et al. 1998, Kanki et al. 2004, Matsushima et al. 2004, Otten et al. 2020, Wang et al. 2021. The effects of TFAM overexpression on mtDNA levels are less clear. Various studies in cell culture demonstrate that TFAM overexpression can be both sufficient (Kanki et al. 2004, Matsushima et al. 2004, and insufficient (Maniura-Weber et al. 2004) to drive increases in mtDNA. The situation in vivo is similar, as two independent studies in Drosophila saw no net effect of TFAM overexpression on mtDNA levels (Matsuda et al. 2013, Cagin et al. 2015, whereas TFAM overexpression in mice was sufficient to drive mtDNA expansion above normal levels (Ekstrand et al. 2004). Previously, we and others showed that global reduction in TFAM activity has a profound negative impact on mtDNA levels in C. elegans, as expected (Sumitani et al. 2011, Lin et al. 2016, Schwartz et al. 2022). Here we ask the converse: is germline overexpression of the worm TFAM homolog, hmg-5, sufficient to increase mtDNA levels in vivo?
To overexpress TFAM (encoded by the hmg-5 gene) in the germ line, we used regulatory elements from the endogenous glh-1 gene, which encodes a highly expressed germline-specific protein (Marnik et al. 2019, Goudeau et al. 2021. We employed two strategies to express untagged TFAM at the glh-1 locus: the viral 2A self-cleaving peptide system, which results in the production of two peptides via ribosomal skipping during translation (Fig. 1A, left); and the C. elegans SL2 trans-splicing recognition element (derived from the operonic gene rla-1), which causes the nascent transcript to be spliced into two independently translated mRNAs (Fig. 1A, right) (Nance and Frokjaer-Jensen 2019). We used CRIPSR/Cas9 genome engineering to insert either T2A::TFAM or SL2::TFAM at the 3' end of the endogenous glh-1 protein coding sequence (Fig.  1A). To avoid known loss of functionality due to the presence of C-terminal tags on TFAM (Schwartz et al. 2022), we expressed TFAM untagged. Though we did not directly test for an increase of TFAM mRNA or TFAM protein, we presumed overexpression based on results below.
To determine the effect of overexpressing TFAM on mtDNA levels, we measured total mtDNA in whole L4 larvae by quantitative PCR (qPCR), as ~90% of total C. elegans mtDNA is derived from the germline Lemire 2002, Bratic et al. 2009). Both means of overexpressing TFAM resulted in a significant increase in mtDNA levels (~25-30%) (Fig. 1B). Conversely, as reported previously (Schwartz et al. 2022), we found that animals homozygous for a reduction-of-function GFP-tagged TFAM/hmg-5 allele, here referred to as TFAM-GFP (rf), had severe mtDNA copy number defects in L4 larvae (Fig. 1B). This defect in mtDNA number could be rescued by overexpressing TFAM in the TFAM-GFP (rf) background (Fig.  1B). Together, these results demonstrate that excess TFAM is sufficient to drive an increase in mtDNA levels in the C. elegans germ line.
We next sought to determine if increasing TFAM activity affected mtDNA quality. To accomplish this, we used a strain containing the uaDf5 mtDNA deletion, which removes 3.1kb of the 13.8kb mitochondrial genome . uaDf5 deletes essential genes and therefore must exist in heteroplasmy with complementing wild-type mtDNA. Strikingly, uaDf5 mutant mtDNAs persist stably over many generations due to the preferential replication of uaDf5 mtDNAs over wildtype mtDNA genomes , Gitschlag et al. 2020, Schwartz et al. 2022, Yang et al. 2022). Owing to this selfish replicative advantage, we hypothesized that excess TFAM would increase the proportion of uaDf5 mtDNA relative to wild-type mtDNA. Indeed, TFAM-O/E (T2A); uaDf5 adult animals contained a significantly higher percentage of mutant mtDNAs (~70%) compared to uaDf5 controls (~60%) (Fig. 1C). This finding suggests that increasing mtDNA levels by overexpressing TFAM favors an even further expansion of uaDf5 mutant genomes over wild-type mtDNAs compared to controls expressing normal TFAM levels.

Worm culture and strains
C. elegans strains were maintained at 20°C on nematode growth medium plates seeded with Escherichia coli OP50 as previously described (Brenner 1974). A list of all strains used/generated in the study is available in the Strain Table below.

Mitochondrial DNA quantification
For mtDNA copy number quantification, qPCR was performed exactly as previously described (Schwartz et al. 2022). Briefly, single late-L4 larvae were picked into 5µL of worm lysis buffer [50 mM KCl, 10 mM Tris-HCl (pH 8.0), 2.5 mM MgCl 2 , 0.45% IGEPAL (Sigma I8896), and 200 µg/mL proteinase K (Invitrogen 2530049)] in PCR tubes, flash frozen at -80°C for 15 minutes, and lysed in a thermal cycler at 60°C for 1 hour followed by 15 minutes at 95°C. Larval worm lysates were diluted with 95 µL of nuclease-free water (Invitrogen 4387936), and exactly 8µL of diluted lysate was used in triplicate for qPCR. Oligos targeting the mtDNA gene nd-1  were used. A standard curve using linearized plasmid containing the nd-1 sequence was run for absolute quantification, and qPCR was performed using BioRad 2X SsoAdvanced Universal SYBR Green Supermix (BioRad 1725271) in a Roche LightCycler 480 machine as previously described (Schwartz et al. 2022).
For uaDf5 heteroplasmy measurement, 60-100 whole adult animals were pooled in 60-100µL of worm lysis buffer in a screw cap 1.5mL microfuge tube, flash frozen at -80°C for 15 minutes, and lysed in a heating block at 60°C for 1 hour followed by 15 minutes at 95°C. Adult worm lysates were diluted 1000X and droplet digital PCR (ddPCR) quantification of uaDf5 and WT mtDNA was performed exactly as previously described (Schwartz et al. 2022).

Statistical analysis and reproducibility
All statistical analysis was performed using GraphPad Prism 9 software. For all data, unpaired two-tailed Student's t-tests were performed, and where applicable no corrections for multiple comparisons were made to avoid type II errors (Armstrong 2014). Data in graphs are shown as Superplots (Lord et al. 2020). Three biologically independent experiments were performed for all experiments and the arithmetic means of biological replicates were used for statistical analysis.